Process for synthesis of novel cationic amphiphiles containing N-hydroxyalkl group for intracellular delivery of biologically active molecules

ABSTRACT

The present invention provides processes for the synthesis of novel cationic amphiphiles capable of facilitating transport of biologically active molecules into cells wherein the said amphiphiles contain N-hydroxyalkyl group and have at least one hydroxyalkyl group containing 1-3 carbon atoms directly linked to the positively charged nitrogen atom.

FIELD OF THE INVENTION

The present invention relates to processes for preparing novel cationicamphiphiles containing N-hydroxyalkyl group. The invention providesnovel compositions containing the said amphiphiles that are useful tofacilitate transport of biologically active molecules into cells. Thearea of medical science that is likely to benefit most from the presentinvention is gene therapy.

BACKGROUND

In gene therapy, patients carrying identified defective genes aresupplemented with the copies of the corresponding normal genes. However,genes (DNA), the polyanionic macromolecules and the cell surfaces of thebiological membranes both being negatively charged, spontaneous entry ofnormal copies of genes into the target cells of patients is aninefficient process (because of electrostatic repulsion). This is whythe past decade has witnessed an unprecedented upsurge of globalinterest in developing efficient gene delivery reagents for introducingnormal genes into the target cells of patients suffering from variousgenetic (inherited) diseases such as cystic fibrosis, Gaucher's illness,Fabry's disease etc. Many gene delivery reagents (also known astransfection vectors) including retrovirus, adenovirus, and cationicamphiphilic compounds (i.e. compounds containing both polar andnon-polar functionalities) are being used as the carriers of polyanionicgenes in combating hereditary diseases in gene therapy. The amphiphilicnature (presence of both polar and non-polar regions in the molecularstructures) of the compounds designed to deliver therapeutically activesmolecules, ensures smooth interaction of these carrier molecules withthe polar and non-polar regions of plasma membranes, compartments withinthe cells and the biologically active molecules itself. At physiologicalpH, the cationic amphiphiles in the form of liposomes or micellesassociate favorably with the negatively charged regions of themacromolecular polyanionic DNA enhancing the intracelluar uptake of theresulting complex between the cationic lipids and the negatively chargedDNA. Reproducibility, high degree of targetability and low cellulartoxicity are increasingly making the cationic amphiphiles thetransfection vectors of choice in gene therapy.

PRIOR ART REFERENCES

An impressive number of cationic lipids with varying structures havebeen reported for the intracellular delivery of therapeutically activemolecules as exemplified by the following references:

Felgner et al., Proc. Natl. Acad. Sci. U.S.A., 84, 7413-7417 (1987),reported the first use of a highly efficient cationic lipidN-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethyl ammonium chloride (DOTMA)as the DNA transfection vector.

U.S. Pat. Nos. 4,897,355 and 4,946,787 (1990) reported the synthesis anduse of N-[.omega..(.omega.-1)dialkyloxy]- andN-[..omega..(.omega.-1)-dialkenyloxy]-alk-1-yl-N,N,N-tetrasubstitutedammonium amphiphiles and their pharmaceutical formulations as efficienttransfection vectors.

Leventis, R. and Silvius, J. R. Biochim. Biophys. Acta 1023, 124-132,(1990) reported the interactions of mammalian cells with lipiddispersions containing novel metabolizable cationic amphiphiles.

U.S. Pat. No. 5,264,618 (1993) reported the synthesis and use ofadditional series of highly efficient cationic lipids for intracellulardelivery of biologically active molecules.

Felgner et al. J. Biol. Chem. 269, 2550-2561 (1994) reported enhancedgene delivery and mechanistic studies with a novel series of cationiclipid formulations.

U.S. Pat. No. 5,283,185 (1994) reported the synthesis and use of3β[N-(N¹,N¹-dimethylaminoethane)carbamoyl]cholesterol, termed as“DC-Chol” for delivery of a plasmid carrying a gene for chloraniphenicolacetyl transferase into cultured mammalian cells.

U.S. Pat. No. 5,283,185 (1994) reported the use ofN-[2-[[2,5-bis[(3-aminopropyl)amino]-1-Oxopentyl]aminoethyl]-N,N-dimethyl-2,3-bis-(9-Octadecenyloxy)-1-Propanaminiumtetra (trifluoroacetate), one of the most widely used cationic lipids ingene delivery. The pharmaceutical formulation containing this cationiclipid is sold commercially under the trade name “Lipofectamine”.

Solodin et al. Biochemistry 34, 13537-13544, (1995) reported a novelseries of amphiphilic imidazolinium compounds for in vitro and in vivogene delivery.

Wheeler et al. Proc. Natl. Acad.Sci. U.S.A. 93, 11454-11459, (1996)reported a novel cationic lipid that greatly enhances plasmid DNAdelivery and expression in mouse lung.

U.S. Pat. No. 5,527,928 (1996) reported the synthesis and the use ofN,N,N,N-tetramethyl-N,N-bis(hydroxyethyl)-2,3-di(oleolyoxy)-1,4-butanediammonium iodide i.e. pharmaceuticalformulation as transfection vector.

7 U.S. Pat. No. 5,698,721 (1997) reported the synthesis and use of alkylO-phosphate esters of diacylphosphate compounds such asphosphatidylcholine or phosphatidylethanolamine for intracellulardelivery of macromolecules.

U.S. Pat. Nos. 5,661,018; 5,686,620 and 5,688,958 (1997) disclosed anovel class of cationic phospholipids containing phosphotriesterderivatives of phosphoglycerides and sphingolipids efficient in thelipofection of nucleic acids.

U.S. Pat. No. 5,614,503 (1997) reported the synthesis and use of anamphipathic transporter for delivery of nucleic acid into cells,comprising an essentially nontoxic, biodegradable cationic compoundhaving a cationic polyamine head group capable of binding a nucleic acidand a cholesterol lipid tail capable of associating with a cellularmembrane.

U.S. Pat. No. 5,705,693 (1998) disclosed the method of preparation anduse of new cationic lipids and intermediates in their synthesis that areusefull for trasfecting nucleic acids or peptides into prokaryotic oreukaryotic cells. These lipids comprise one or two substituted arginine,lysine or ornithine residues, or derivatives thereof, linked to alipophilic moiety.

U.S. Pat. No. 5,719,131 (1998) has reported the synthesis of a series ofnovel cationic amphiphiles that facilitate transport of genes intocells. The amphiphiles contain lipophilic groups derived from steroids,from mono or dialkylamines, alkylamines or polyalkylamines.

Although the above mentioned cationic lipids have been successfullyexploited for the intracellular delivery of genes, the efficiencies forthe intracellular uptake procedures are insufficient and need to beimproved. The transfection activities of most of the above mentionedcationic lipids are modest and therefore substantial quantities of thesecationic lipids must be consumed. The associated cellular toxicities ofthe lipids and the metabolites thereof are, thus naturally, issues ofconcern. Accordingly, demands for developing new class of cationicamphiphiles with high transfection efficiencies and low cellulartoxicities continue in this field of art.

OBJECTS OF THE INVENTION

The main objective of the present invention is to provide novel simpleand economic processes for the synthesis of cationic amphiphiliccompounds containing non-toxic N-hydroyalkyl group.

Another objective of the invention is to provide processes for thesynthesis of said novel cationic amphiphilic compounds which are usefulfor delivery of therapeutically effective amounts of biologically activemolecules into cells/tissues of patients.

Yet another objective of the invention is to provide novel processes forthe synthesis of cationic amphiphilic compounds such that a hydrophobicgroup is either directly linked to the positively charged Nitrogen atomor is linked to the said Nitrogen atom via an ester or methylene group.

Still another objective of the invention is to provide novel processesfor the synthesis of cationic amphiphilic compounds with at least onehydroxyalkyl group containing 1-3 carbon atoms directly linked to thepositively charged Nitrogen atom.

Yet another objective of the invention is to provide novel cationicamphiphilic compounds without any glycerol backbone in their structure.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to novel cationic amphiphiles containingnon-toxic N-hydroxyalkyl group and provides processes for thepreparation said amphiphilic compounds. The novel cationic amphiphilescontaining N-hydroxyalkyl group of this invention are potentially usefulto deliver into the cells of patients therapeutically effective amountsof biologically active molecules. The area of medical science that islikely to benefit most from the present invention is gene therapy.

Cationic amphiphiles disclosed in the present invention possess severalnovel structural features. These features may be compared with cationicamphiphilic structures disclosed in Felgner et al. J. Biol. Chem., 269,2550-2561 (1994), a representative structure of which is1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide(“DMRIE”) and to those disclosed by Bennett et al. J. Med. Chem., 40,4069-4078 (1997), a representative structure of which isN,N-[Bis(2-hydroxyethyl)]-N-methyl-N-[2,3-bis(tetradecanoyloxy)propyl]ammonium chloride (“DMDBP”).

The following distinctive structural features are common to the cationicamphiphiles disclosed in the present invention: (1) the presence of ahydrophobic group which is either directly linked to the positivelycharged nitrogen atom or is linked to the positively charged nitrogenvia an ester group, (2) the presence of at least one hydroxyalkyl groupcontaining 1-3 carbon atom that is directly linked to the positivelycharged nitrogen atom and (3) unlike many other glycerol-based cationicamphiphiles, the cationic transfection lipids disclosed in the presentinvention do not have any glycerol backbone in their moleculararchitecture. It is believed that these unique structural featurescontribute significantly to the increased transfection efficiencies ofthe cationic amphiphiles disclosed herein. The enhanced in vitrotransfection efficiencies ofN,N,-di-[O-hexadecanoyl]hydroxyethyl-N-hydroxyethyl-N-methylammoniumbromide (DOHEMAB) and N,N-di-n-hexadecyl-N,N-dihydroxyethylammoniumbromide (DHDEAB), the two novel transfection lipids of the presentinvention, is compared to Lipofectamine, the most widely usedcommercially available highly efficient transfection lipid.

According to the practice of the present invention, “cationic” means thepositive charge is either on a quaternized nitrogen atom or on aprotonated tertiary nitrogen atom. The cationic characters of theamphiphiles may contribute to the enhanced interaction of theamphiphiles with biologically active molecules (such as nucleic acids)and/or with cell constituents such as plasma membrane glycoproteins.Such enhanced interaction between the cationic transfection lipid andthe biological by active molecules and/or cell membrane constituents mayplay a key role in successfully transporting the therapeutic moleculesinto the cells.

The cationic amphiphiles of the present invention have certain commonstructural and functional groups. As such, the said cationic amphiphiliccompounds may be represented by the following generic formula:

Wherein

n is an integer between 1 and 3,

R₁ may be H, saturated or unsaturated aliphatic group (C₈-C₂₀) or longchain saturated or unsaturated alkyl group (C₇-C₁₉).

R₃ may be hydroxyalkyl group of 1-3 carbon atoms,

R₂ may be a long chain saturated alkyl group (C₇-C₁₉) or[(CH₂)_(n)-Z-R₂]

Z may be methylene group (—CH₂), or an esteric group (—O—CO—)

Various novel amphiphilic compounds having the above basic structuralformula are described in co-pending U.S. patent application Ser. No........... (Indian Application No.3324/DEL/98,3325/DEL/98 and 3327/DEL98all dated Nov. 9, 1998). Further, in the said amphiphiles the preferenceof C₈-C₂₀ is specifically low because below the chain length of C₈ theamphiphiles loose the aggregation property and the specific reason fornot using carbon chains longer than C₂₀ is because they are notcompatible with biological membranes in terms of their lengths. Further,the additional linking groups that may be practised within the scope ofthis invention include —O—(ether) CONH-amide) group etc. Among the threedifferent linking groups described in the invention herein below, theesteric group is most prefeiri

Further, the products obtained during the synthesis of the saidamphiphilic compounds are hydrolysis products. The molecules were soconstructed to aid in their breakdown within a cell subsequent toperforming the task of transfection. The structure of the amphiphilesmay also be altered by a combination of alkyl and amine moieties, whichstructures would fall within the teachings and scope of the presentinvention. Such modifications are apparent to those skilled in the art.

Accordingly, the invention provides cationic amphiphiles represented bythe following structural formula (I):

Wherein:

n is an integer between 1 and 3;

R₁ represents either H or a saturated aliphatic group;

R₂ independently represents a long chain saturated alkyl group (from C₇to C₁₉);

R₃ is a small hydroxyalkyl group containing 1-3 carbon atoms;

X is either a halogen atom or a tosylate group.

Z represents a methylene (—CH₂—) group;

The process for the synthesis of said cationic amphiphilic compoundshaving the above structural formula (I) comprises reacting the secondaryamine containing the N-hydroxyalkyl group such as diethanolamine with asaturated alkyl halides or saturated alkyl tosylates in a polar solventin the presence of a weak tertiary base.

In an embodiment of the invention, the N-hydroxyalkyl group may bediethanolarnine.

In another embodiment, the saturated alkyl bromides employed in theprocess may be selected from the group consisting of n-hexadecylbromide.

In yet another embodiment, the polar solvents in which the reaction maybe carried out may be selected from the group comprising alcohols,diemethyl formamide, dimethylacetamide, acetonitrile, methylisobutylketone.

In still another embodiment of the invention, the base may be selectedfrom sodium or potassium carbonate.

In yet another embodiment, the reaction for the synthesis of saidamphiphilic lipids may be carried out at a temperature in the range of0°-250° C., preferably at the reflux temperature (boiling point of thesolvent selected).

According to an aspect of the invention, a pair of particularlyeffective representative cationic amphiphiles that may be synthesised bythe said reaction, would includeN,N-di-n-hexadecyl-N,N-dihydroxyethylammonium bromide (DHDEAB),amphiphile No 1 and N-n-hexadecyl-N,N-dihydroxyethylammonium bromide(HDEAB), amphiphile No. 2 represented as under:

The process for the synthesis of said cationic lipids is schematicallyrepresented hereunder.

The invention further provides cationic amphiphilic compoundsrepresented by the structural formula (II):

wherein:

n is an integer between 1 and 3;

R₁ independently, represents either a saturated aliphatic group or anunsaturated aliphatic group (from C₈ to C₂₀);

Z represents a methylene (—CH₂—) group;

R₂, independently, represents a long-chain saturated alkyl group (fromC₇ to C₁₉);

R₃ is a small alkyl group (from C₁ to C₃);

X is either a halogen atom or a tosylate group.

The process for the synthesis of said cationic amphiphilic compoundshaving the above structural formula,comprises:

(a) coupling the aliphatic unsaturated aldehyde with the alkyl aminefollowed by the reduction of the resulting imine to obtain thecorresponding secondary amine;

(b) reacting the secondary amine obtained in step (a) with thehydroxyl-protected hydroxyalkyl halide followed by removal of thehydroxyl protecting group to obtain the corresponding N-hydroxyalkylgroup containing tertiary amine and

(c) quaternizing the resulting N-hydroxyalkyl group containing tertiaryamine obtained in step (b) with an alkyl halide or alkyl tosylates in amixed polar solvent.

In an embodiment of the invention, the saturated or unsaturated aldehydeused may be oleyl aldehyde.

In another embodiment, the alkyl amine used may be n-octadecyl amine.

In yet another embodiment, the tertiary amine may be quarternized withalkyl iodide selected from methyl iodide.

In yet another embodiment, the mixed polar solvent may comprise amixture of methanol and chloroform.

In yet another embodiment, the resultant tertiary amine of step (b) maybe N-hydroxyethyl-N- oleyl-N-n-octadecyl-amine

In an embodiment, the reaction of amine with the aldehyde in step (a) isgenerally performed in a dry chlorinated solvent such as dichloromethanewith temperature in the range of −78° C. to 10° C. and the reduction ofthe resulting intermediate imine is carried out with sodium borohydridein a mixed polar solvent such as methanol and dichloromethane withtemperature in the range of −5° C. to 40° C. The step (b) is generallycarried out in a polar solvent such as ethyl acetate, N,N-dimethylformamide, acetonitrile and the like in the presence of a weak base suchas sodium or potassium carbonate. The solvent selected in removing thehydroxyl protecting group in step (b) is generally polar aprotic innature such as tetrahydrofuran, dimethyl formamide and the like. Thefinal quarternization in step (c) is carried out using the appropriatealkyl polar solvent such as methanol and chloroform at a temperature inthe range −5° C. to 40° C.

According to this aspect of the invention, a particularly effectiverepresentative pair of cationic transfection lipids prepared by theabove process would include, for example,N-methyl-N-n-octadecyl-N-oleyl-N-hydroxyethyl-ammonium chloride(MOOHAC), amphiphile No 3 andNN-di-n-octadecyl-N-methyl-N-dihydroxyethylammonium chloride (DOMBAC),amphiphile No. 4, whose structural formulae are represented hereunder:

A schematic representation of the process described above for thesynthesis of said cationic amphiphiles is provided hereunder forreference:

The invention further provides a process for the preparation of cationicamphiphiles represented by the structural formula (mH) given hereunder:

wherein:

n is an integer between 1 and 3, Z an ester group (—O—CO—) and R₁ and R₂independently, represent a long-chain saturated or unsaturated alkylgroup (from C₇ to C₁₉), R₃ is a small alkyl group (C₁ to C₃) and X iseither a halogen atom or a tosylate group.

The process for the synthesis of said cationic amphiphilic compounds,comprises:

(a) reacting an acid chloride with a tertiary amine containing theN,N-dihydroxyalkyl group to obtain the hydrochloride salt of thecorresponding di-Oacylated product,

(b) neutralizing the resulting hydrochloride salt obtained in step (a)with alkali and

(c) quaternizing the resulting tertiary amine obtained in step (b) withthe appropriate hydroxy-alkyl halide.

In an embodiment of the invention, the acid chloride used in step (a)may be n-hexadecanoyl chloride.

In another embodiment, the tertiary amine in step (a) may beN-methyldiethanolamine.

In yet another embodiment, the reaction in step (a) may be performed ata temperature in the range of 10°-50° C., in a polar aprotic solventselected from the group comprising N,N-dimethylformamide, acetonitrileand the like.

In yet another embodiment, the neutralization in step (b) may be carriedout in a biphasic solvent such as mixture of dichloromethane and wateror ethyl acetate and water using strong alkali such as NaOH or KOH.

In yet another embodiment, the quarternization in step (c) is carriedout at a temperature in the range of 40° C. to 100° C. depending on thenature of alkyl halides used.

In another embodiment, the hydroxyalkyl halide used in step (a) is2-bromoethanol.

According to this aspect of the invention, a particularly effectivecationic transfection lipid that may be prepared by the above processincludes-N,N,-di[O-hexadecanoyl]hydroxyethyl-N-hydroxyethyl-N-methylammoniumbromide (DOHEMAB), amphiphile 5 having the structural formula ashereunder:

The said process taught for the synthesis of cationic lipids isschematically represented hereunder:

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1 and 2 provide the in vitro cellular toxicities of the cationicamphiphiles disclosed in the present invention.

FIGS. 3-7 provide in vitro transfection efficiencies for the cationicamphiphiles disclosed in the present invention and that ofLipofectamine, the most widely used commercially available transfectionlipid, under certain conditions.

Formulations Containing the Cationic Amphiphiles of the Invention andthe in vitro Administration Thereof

The present invention provides for various formulations that facilitateintracellular delivery of biologically active molecules. Theformulations of the transfection lipids disclosed herein were preparedeither by keeping the amount of the cationic amphiphiles constant at 1mM and varying the amounts of the auxiliary neutral lipid (cholesterol)within the range 0.2 mM to 1 mM or by adding varying amounts of thenovel single-chain auxiliary cationic lipidN-n-hexadecyl-N,N-dihydroxyethylammonium bromide (HDEAB) within therange of 0.15 mM-0.5 mM to a mixture containing 1 mM of cholesterol and1 mM twin-chain novel cationic amphiphiles disclosed herein. As shown inFIG. 3 of the drawings accompanying this specification, combination of 1mM DHDEAB and 0.6 mM Cholesterol is a highly efficient formulation.Similarly, FIG. 4 of the drawings accompanying this specification showsan additional highly effective formulation containing 0.2 mM HDEAB, 1 mMDHEAB and 1 mM Cholesterol.

Cellular Toxicities of the Cationic Amphiphiles Disclosed in theInvention

The viabilities of cells in presence of various cationic amphiphilesdisclosed herein were checked according to the standard protocoldescribed in “Animal Cell Culture. 2nd Edition. Ed. I.R.L Press t,Oxford University Press (1997)”. The transfection efficiencies of thecationic lipids were studied in the range of 0-10 nmole and within thislimit, the cell toxicities were observed to be minimal and the cellviabilities were determined to be more than 80% as summarized in FIGS. 1and 2. The cationic amphiphiles were used at a concentration of 1 mMtogether with 1 mM cholesterol as the neutral co-lipid.

Main Advantages of the Present Invention

1. The procedures for making these novel cationic amphiphiles beingsimple, the prices of this new transfection reagent should be remarkablylower than that of LIPOFECTAMINE™ and LIPOFECTIN™

2. The transfection efficiency of the novel cationic amphiphiles aremuch better than that of LIPOFECTIN™ and comparable to or better thanthe transfection efficiencies of LIPOFECTAMINE™.

3. The cationic amphiphiles disclosed in the present invention have beentested positive for transfecting COS 1, Hela, Vero, CV 1 and NIH3T3cells and the primary cell line, Rat Skin Fibroblasts.

The following examples are given by way of illustration of the presentinvention and therefore should not be construed to limit the scope ofthe present invention.

EXAMPLE 1

Cell Transfection Assay

Separate 1 μmole (80 μl of a 0.0126 M stock solution in chloroform) ofN,N-di-n-hexadecyl-N,N-dihydroxyethylammonium bromide (DHDEAB) and 1μmole of the neutral colipid cholesterol (40 μl of a 0.0255 M stocksolution in chloroform) were combined in a glass vial. The solvent wasevaporated initially by a thin flow of moisture free nitrogen and thenunder high flow for two minutes and then further dried under vacuum (1mm Hg) for 6 hours.

The dried lipid film was then hydrated with sterile deionized water (1ml) overnight (9-10 hours) in room temperature to produce a dispersedsuspension. The hydrated mixture was then vortexed for 1 minute and wassonicated in a probe sonicator at a constant 25 W sonic power for 5minutes. The concentrations of both DHDEAB and cholesterol in theresulting solution obtained after sonication was 1 mM.

For preparation of the transfecting solution, DNA encodingβ-galactosidase (pCH 110 construct as described in Borras et al.Developmental Biology 127, 209-219, 1988) was dissolved in DMEM culturemedium (GIBCO/BRL No. 12800-017). The resulting solution had a DNAconcentration of 454 μM (assuming an average molecular weight of 660Dalton per base pair for nucleotides in the encoding DNA).

A 24 μl aliquot of the sonicated dispersion obtained (containing 1 mM ofboth cationic lipid and the colipid) was pipetted into separate wells of96-well plate already containing 51 μl DMEM solution. The resulting 330μM solution (with respect to both the cationic amphiphile and thecolipid) was then three fold serially diluted for five times to obtaindiluted amphiphile and cholesterol solutions within the range of 330μM-4 μM, the volume of each resulting diluted solution being 50 μl. Toeach 50 μl resulting diluted solution, 2 μl of 454 μM DNA solution wasadded and the mixture was kept stirring for {fraction (1/2+L )} hour atroom temperature to allow for the DNA-lipid complexation.

A COS-1 (SV 40 transformed African green monkey kidney, # ATCCCRL 1650,from ATCL, Maryland, U.S.A.) cell line was used for the in vitro assay.The cells were cultured in DDEM media containing 10% fetal bovine serum(“FBS”, Sigma F 0392) supplemented with Penicillin, Streptomycin andKanamycin. Cells were plated into 24-well tissue culture plate to adensity of approximately 5-6×10⁴ cells/well and kept for about 12-14hours until a near confluent (75-80%) pattern had been achieved.

To the DNA-lipid complex volume of 52 μl, 350 μl of DMEM was added andmixed thoroughly. The 24-well plates with COS-1 cells were aspirated inorder to remove growth medium. The cells were washed once with phosphatebuffered saline (PBS). Now to consecutive wells of 24-well plates, 201μl of the DNA-lipid complex was added over the cells and the mixture wasincubated for 3 hours in a CO₂ incubator. 200 μl of 10% FBS containingDMEM was added to each cell well. After a further 20 hour incubationperiod, the medium was removed and a fresh DMEM with 10% FBS was added.Following a further 24 hours incubation period, cells were assayed forexpression of β-galactosidase and protein.

For protein estimation, the resultant medium was removed from the platesand the cells were were washed with PBS. Lysis buffer (100 μl containing250 mM Tris.HCl, pH 8.0, 0.5% NP40) was added and the cells were lysedfor 30 minutes. Lysate was passed through the pipette tip several timesto dislodge and dissolve the cell fragments, 2.5 μl of lysate from eachwell was transferred to a 96-well plate containing 2.5 μl H₂O, 25 μlreagent A and 200 μl reagent B from the detergent compatible Bio-Radprotein assay kit (Bio-Rad 500-0116). The plates were read in an Elisareader (Molecular Devices, Vmax) with a 650 nm filter. Proteins standardcurve was also constructed by the same procedure using bovine serumalbumin as standard.

The level of β-galactosidase activity in a 50 μl lysate was measured byadding to 50 μl of 2×β-gal mixture containing 1.33 mg/mlo-nitrophenyl-β-D-galactopyranoside (Sigma N1127), 200 mM sodiumphosphate pH 7.15 and 200 mM magnesium sulfate. Theo-nitrophenyl-β-D-galactopyranoside, following enzymatic hydrolysis at37 ° C. gave a yellow colour which was detected and measured by a platereader equipped with a 405 mn filter. A β-galactosidase (Sigma No.G6512) standard calibration graph was constructed for calculating theamount of β-galactosidase in each well. Following subtraction ofbackground readings, optical data determined by the plate reader alloweddetermination of β-galactosidase activity and protein content for eachwell. The results were finally summarized in terms of microunit ofβ-galactosidase per microgram of protein content (Reporter geneactivity). Representative examples of such reporter gene activitiesobserved with the cationic amphiphiles of the present invention areshown in FIGS. 2-4 of the drawings accompanying this specification.

The above described example is only an illustration with pCH 110. Wehave performed similar experiments with pCMV β-gal (GIBCO BRL,Gaithersberg, U.S.A.) & pGFPN1 (Clone Tech, Palo Alto, U.S.A.) and wehave obtained similar reporter gene activities.

EXAMPLE 2

Procedure for the simultaneous preparation ofN,N-di-n-hexadecyl-N,N-dihydroxyethylammonium bromide (DHDEAB),amphiphile No 1 and N-n-hexadecyl-NN-dihydroxyethylammonium bromide(HDEAB), amphiphile No. 2.

A mixture of diethanolamine (1 g, 9.5 mmol), n-hexadecyl bromide (2.9 g,9.5 mmol) and potassium carbonate (1.44 g, 10.5 mmol) was Tefluxed inmethanol for 48 hours. Methanol was removed on a rotatory evaporator andthe residue was extracted with chloroform (3×100 ml). The combinedchloroform extract was filtered repeatedly (3 times) to remove potassiumcarbonate. Chloroform was removed from the filtrate on a rotaryevaporator and the residue was taken in 50 ml 80:15:5 (v/v)acetonitrile:ethyl acetate:methanol. One equivalent of n-hexadecylbromide (2.9 g, 9.5 mmol) was added and the mixture was refluxed for 56hours. At this point, an additional equivalent of potassium carbonate(1.44 g, 10.5 mmol) was added and refluxing was continued for another 30hours. Finally, one more equivalent of n-hexadecyl bromide (2.9 g, 9.5mmol) was added and the mixture was refluxed for 24 hours. The solventwas removed on a rotary evaporator and the residue was extracted withchloroform (100 ml). Potassium carbonate was filtered from thechloroform extract and the chloroform was removed on a rotaryevaporator. The residue was dissolved in ethyl acetate (30 ml) with thehelp of little methanol (0.3-0.5 ml) and the resulting solution of theproduct mixture showed three spots (with R_(f)=0.9, 0.8 and 0.7) on thinlayer chromatography (TLC) using 30:70 (v/v) methanol:chloroform as theTLC developing solvent. The solution was kept at 4° C. for 36 hours. Theprecipitate that appeared at this point was predominantly the compoundwith R_(f)=0.9 which was further purified by silica gel chromatographyusing finer than 200 mesh size silica and eluting with 2-5% methanolic(by volume) chloroform. The NMR and HRMS of this column purified product(yield: 470 mg) showed it to be a O-n-hexadecyl derivative of the titleamphiphile No 1 that did not show any gene transfection activity. Themother liquor was concentrated and dry packed with 60-120 mesh sizesilica. The dry packed residue was loaded on a 60-120 mesh size silicacolumn and was eluted first with ethyl acetate and then with chloroformto wash off unreacted n-hexadecyl bromide and diethanolamine. Thecompounds with R_(f)=0.8 and 0.7 were then isolated as a mixture bychanging the eluent to 90:10 (v/v) chloroform:methanol. The isolatedproduct mixture was finally dry packed with finer than 200 mesh sizesilica and the dry packed mixture was loaded on a finer than 200 meshsize silica column. The title amphiphiles 1 and 2 (with R_(f)=0.8 and0.7 respectively) were finally isolated in pure forms by eluting thedry-packed column carefully with chloroform containing 2-5% methanol (byvolume). The yields of the purified amphiphile 1 and amphiphile 2 wererespectively 8% (500 mg, 0.787 mmol) and 12.7% (500 mg, 1.2 mmol).

¹H-NMR of amphiphile 1 (200 MHz, CDCl₃): δ/ppm=0.9 [t, 6H, CH₃—(CH₂)_(n)—]; 1.20-1.5 [m, 52H, —(CH ₂)₁₃—]; 155-1.85 [m, 4H,(HOCH₂—CH₂)₂N⁺(CH₂—CH ₂—)₂]; 3.4-3.6 [m, 4H, (HOCH₂—CH₂)N⁺(CH ₂—CH₂—)₂];3.65-3.80 [m, 4H, (HOCH₂—CH ₂)₂N⁺(CH₂—CH₂—)₂]; 4.05-4.2 [m, 4H, (HOCH₂—CH₂)₂N⁺(CH₂—CH₂—)₂]; 4.75-4.95(m, 2H, —OH).

HRMS (FABS) m/z: Calcd (for C₃₆H₇₆NO₂ the 4°-ammonium ion) 554.5876found 554.5899

¹H-NMR of amphiphile 2 (200 MHz,CDCl₃): δ/ppm=0.9 [t, 3H, CH₃—(CH₂)_(n)—]; 1.15-1.5 [m, 26H, CH₃—(CH ₂)₁₃—]; 1.75-1.85 [m, 2H,(HOCH₂—CH₂)₂N⁺(CH₂—CH ₂—)₂—]; 3.2-3.35 [m, 2H, (HOCH₂—CH₂)₂N⁺(CH₂—CH₂—)₂]; 3.35-3.5 [m, 411, (HOCH₂—CH ₂)₂N⁺(CH₂—CH₂—)₂]; 4.04.15 [m,4H, (HOCH ₂—CH₂)₂N⁺(CH₂—CH₂—)₂];

HRMS (FABS) m/z : Calcd (for C₂₀H₄₄NO₂ the 4°-ammonium ion) 330.3372found 330.3346.

EXAMPLE 3

Procedure for preparingN-methyl-N-n-octadecyl-N-oleyl-N-hydroxyethyl-ammonium chloride(MOORAC), amphiphile No 3.

Step (a). 100 ml dry dichloromethane was cooled to 0° C. and to the colddichloromethane solution was added 1.9 g of oleyl aldehyde (7.14 mmol),1.92 g of stearyl amine (7.14 mmol) and 10.9 g of anhydride magnesiumsulfate (7.14 mmol). The mixture was kept under stirring for 3 hourswhile the temperature of the stirred mixture raised gradually from 0° C.to room temperature. The magnesium sulfate was filtered from thereaction mixture and the filtrate was diluted with 50 ml of methanol.The diluted dichloromethane/methanol solution was cooled to 0° C. and tothe cold solution, 0.54 g Sodium borohydride (14.0 mmol) was added. Thesolution was kept stirred for 4 hours during which time the temperatureof the reaction mixture gradually raised to room temperature. Thereaction mixture was taken in 100 ml chloroform, washed with water(2×100 ml), the chloroform layer was dried over anhydride magnesiumsulfate and filtered. Chloroform was removed from the filtrate on arotary evaporator and column chromatographic (using 60-120 mesh sizesilica) purification of the residue using 20-50% ethyl acetate inpet-ether as the eluent afforded 2.34 g (64% yield) of the desiredintermediate secondary amine, namely, N-oley-N-n-octadecylamine.

Step (b). A mixture of 0.95 g (1.83 mmol) of N-oleyl-N-n-octadecylamineobtained in step (a) and 0.67 g (1.83 mmol) of2-bromoethyl-tertabutyl-diphenylsilyl ether [prepared conventionally bythe reaction between 2-bromoethanol and tetrabutyl-diphenylsilylchloride in the presence of triethylamine and N,N-dimethylaminopyridine] was refluxed in ethyl acetate in presence of anhydrouspotassium carbonate (0.28 g, 2.01 mmol) for 48 hours. The reactionmixture was taken in 100 ml chloroform, washed with water (2×100 ml),dried over anhydrous magnesium sulfate and filtered. Chloroform wasremoved from the filtrate on a rotary evaporator. Silica gel columnchromatographic purification of the resulting residue using 60-120 meshsize silica and 3-4% ethyl acetate (by volume) in pet-ether as theeluent afforded 0.69 g (47% yield) of the intermediate tertiary amine,namely the O-tetrabutyl-diphenylsilyl derivative ofN-2-hydroxyethyl-N-oleyl-N-n-octadecylamine. Thetert-butyl-diphenylsilyl protecting group of this intermediate tertiaryamine (0.67 g, 0.84 mmol) was conventionally removed by stirring withtetrabutylammonium fluoride (2.1 mmol, 2.1 ml of 1M tetrabutylammoniumfluoride solution in tetrahydrofuran) in dry tetrahydrofuran (cooled to0° C.) for 4 hours during which the temperature of the reaction mixturegradually raised to room temperature. The usual work-up and silica gelcolumn purification (as described above in detail for isolating pureprotected intermediate except that the eluent used in this case was7-10% ethyl acetate in pet-ether) of the product mixture afforded 0.32 g(67.5% yield) of pure deprotected tertiary amine namely,N-oley-N-n-octadecyl-N-2-hydroxyethylamine.

Step (c). Quaternization of 0.12 g (0.21 mmol) ofN-oleyl-N-n-octadecyl-N-2-hydroxyethylamine obtained in step (b) wascarried out in 5 ml of 5:4 chloroform methanol (v/v) at room temperaturefor overnight using huge excess of methyl iodide (4 ml). The solventswere removed on a rotary evaporator and silica gel columnchromatographic purification of the residue using 60-120 mesh sizesilica and 3-4% methanol (by volume) in chloroform afforded thequaternary iodide salt of the title A amphiphile. Finally, 0.08 g ofpure title amphiphile No. 3 in 91.9% yield was obtained by subjectingthe quaternized ammonium iodide salt (0.1 g, 0.14 mmol) to repeated (4times) chloride ion-exchange chromatography, each time using a freshlygenerated Amberlyst A-26 chloride ion exchange column column and about80 ml of chloroform as the eluent. All the isolated intermediates gavespectroscopic data in agreement with their assigned structures shown inFIG. 4. Thus, the title amphiphile 3 was prepared in 3 steps with anoverall yield of 18.7%.

¹H-NMR of amphiphile 3 (200 MHz, CDCl₃): δ/ppm=0.89 [t, 6H, CH₃—(CH₂)_(n)—]; 1.20-1.45 [m, 52H, —(CH₂)₁₃—]; 1.60-1.80 [(br, 4H,CH₃(HOCH₂—CH₂)N⁺(CH₂—CH ₂—)₂]; 1.90-2.10 (m, 4H, —CH ₂—CH═CH—CH₂—); 3.35[s, 3H, CH ₃(HOCH₂—CH₂)N⁺(CH₂—CH₂—)₂)]; 3.41-3.58 [br, 4H,CH₃(HOCH₂—CH₂)N⁺(CH ₂—CH₂—)₂]; 3.65-3.80 [br, 2H,CH₃(HOCH₂—CH₂)N⁺(CH₂—CH₂—)₂]; 4.07-4.12 [br, 2H, CH₃(HOCH₂—CH₂)N⁺(CH₂—CH₂—)₂]; 5.32 (m, 2H, —CH₂—CH═CH—CH₂—).

HRMS (FABS) m/z : Calcd (for C₃₉H₈₀NO, the 4°-ammonium ion) 578.6239,found 578.6233.

EXAMPLE 4

Procedure for preparingN,N,-di[O-hexadecanoyl]hydroxyethyl-N-hydroxyethyl-N methylammoniumbromide (DOHEMAB), amphiphile 5.

Step (a). Di-O-palmitoylation of N-methyl-diethanolamine was effectedfollowing a published protocol [Tundo et al. J. Am. Chem. Soc. 104,456-461]. Briefly, N-methyl-diethanolamine (1 g, 8.39 mmol) was reactedwith palmitoyl chloride (5.075 g, 18.5 mmol) in 10 ml dryN,N-dimethylformamide at 0° C. and the temperature was gradually raisedto room temperature within a period of 4 hours. The resultinghydrochloride salt of N-methyl-di-O-palmitoylethanolamine was filtered,crystallized from 20 ml of dry ether. Finally, recrystallization from 20ml 5:15 (v/v) methanol:ethyl acetate afforded 3.8 g of the purehydrochloride intermediate in 68% yield.

Step (b). The recrystallized hydrochloride salt (200 mg) was stirred for5 minutes in a dichloromethane (10 ml)/1.0 M aqueous NaOH (10 ml)biphasic system. The top aqueous layer was discarded and the lowerorganic layer was washed with water (2×50 ml), dried over anhydroussodium sulfate, filtered and dichloromethane was removed on a rotaryevaporator. Pure N-methyl-di-O-palmitoylethanolamine (0.14 g, 0.22 mmol,74.1% yield) was purified from the residue by silica gel columnchromatography using 60-120 mesh size silica and 98:2 (v/v)chloroform:methanol as the eluent.

Step (c). Quaternization of N-methyl-di-O-palmitoylethanolamine waseffected by reacting the purified tertiary amine (0.14 g, 0.22 mmol)with 1.5 equivalent of neat 2-bromoethanol (0.063 g, 0.34 mmol) at 85°C. for 4 hours. The quaternized title amphiphile salt 5 was crystallizedfrom the residue using 10 ml 2:8 (v/v) benzene: n-pentane. Silica gelcolumn chromatographic purification of the resulting crystal using60-120 mesh size silica and 4:96 (v/v) methanol:chloroform as the eluentfinally afforded 0.04 g of the pure title amphiphile 5 in 16.5% yield.All the isolated intermediates gave spectroscopic data in agreement withtheir assigned structures. Thus, the title amphiphile 5 was prepared in3 steps with an overall yield of 8.3%

¹H-NMR of amphiphile 5 (200 MHz, CDCl₃): δ/ppm=0.88 [t, 6H, CH₃—CH₂—C₁₃H₂₆—]; 1.20-1.40 [m, 48H, —(CH ₂)_(n)—]; 1.50-1.68 [m, 4H,CH₃(HOCH₂—CH₂)N⁺(CH₂—CH₂—O—CO—CH ₂—CH₂—)₂]; 2.21-2.40 [2t, 4H, CH₃(HOCH₂13 CH₂)N⁺(CH₂—CH₂—O—CO—CH ₂—CH₂—)₂]; 3.43 [s, 3H, CH₃(HOCH₂—CH₂—CH₂—O—CO—CH₂—CH₂—)₂]; 3.50-4.30 [m, 9H, CH₃(HOCH ₂—CH₂)N⁺(CH ₂—CH₂—O—CO—CH₂—CH₂—)₂]; 4.51-4.58 [br, t, 4H,CH₃(HOCH₂—CH₂)N⁺(CH₂—CH ₂—O—CO—CH₂—CH₂—)₂].

HRMS (FABS) m/z: Calcd (for C₃₉H₇₉NO₅, the 4°-ammonium ion) 641.5958,found 641.5907.

What is claimed is:
 1. A process for the synthesis of novel cationicamphiphiles that facilitate intracellular delivery of biologicallyactive molecules, said amphiphiles represented by the followingstructural formula (I):

wherein: n is an integer between 1 and 3; R₁ independently, representseither a saturated aliphatic group or an unsaturated aliphatic group(from C₈ to C₂₀); Z represents a methylene (—CH₂—) group; R₂,independently, represents a long-chain saturated alkyl group (from C₇ toC₁₉); R₃ is a small allyl group (from C₁ to C₃); X is either a halogenatom or a tosylate group, said process comprising the steps of: (a)coupling an appropriate aliphatic saturated or unsaturated aldehyde withan alkyl amine followed by the reduction of the resulting imine toobtain the corresponding secondary amine; (b) reacting the secondaryamine obtained in step (a) with the appropriate hydroxyl-protectedhydroxyalkyl halide followed by removal of the hydroxyl protecting groupto obtain the corresponding N-hydroxyalkyl group containing tertiaryamine, and (c) quaternizing the resulting N-hydroxyalkyl groupcontaining tertiary amine obtained in step (b) with the appropriatealkyl halide or alkyl tosylates in a mixed polar solvent.
 2. A processas claimed in claim 1 wherein the saturated or unsaturated aldehyde usedis oleyl aldehyde.
 3. A process as claimed in claim 1 wherein the alkylamine used is n-octadecyl amine.
 4. A process as claimed in claim 1wherein the tertiary amine is quarternised with methyl iodide.
 5. Aprocess as claimed in claim 1 wherein the mixed polar solvent in step(c) comprises a mixture of methanol and chloroform.
 6. A process asclaimed in claim 1 wherein the resultant tertiary amine of step (b) isN-hydroxyethyl-N-oleyl-N-n-octadecyl-amine.
 7. A process as claimed inclaim 1 wherein the reaction of amine with aldehyde is performed indichloromethane.
 8. A process as claimed in claim 1 wherein the reactionis carried out at a temperature ranging between −78° C. to 10° C.
 9. Aprocess as claimed in claim 1 wherein the reaction is followed by thereduction of the resulting imine with sodium borohydride in a mixedpolar solvent at a temperature in the range of −5° C. to 40 ° C.
 10. Aprocess as claimed in claim 1 wherein the reaction of step (b) iscarried out in the polar solvent to obtain tertiary intermediate.
 11. Aprocess disclaimed in claim 1 wherein the polar solvent is selected fromfrom the group of ethyl acetate, N,N methylformamide, and acetonitrile.12. A process as claimed in claim 1, wherein the reaction is carried outin the presence of weak base selected from sodium carbonate andpotassium carbonate.
 13. A process as claimed in claim 1 wherein thesolvent used in step (b) for the removal of hydroxyl protecting group isselected from the group consisting of tetrahydrofuran and dimethylformamide.
 14. A process as claimed in claim 1 wherein quarternizationin step (c) is carried out at a temperature ranging between −5° C. to40° C.